Inhibition of RNA and DNA synthesis in Sertoli cells by co-culture with spermatogenic cells

Sertoli cell monolayers were prepared from 30-day-old rat testes and cultured for 7 days to eliminate contaminant germ cells. Some of these monolayers were co-cultured with a spermatogenic cell preparation enriched in pachytene spermatocytes and round spermatids (fraction 3 from a Percoll gradient), isolated from 30-day-old rat testes. After co-culture for 4 to 48 h, germ cells were removed. RNA synthetic activity in rat Sertoli cell cultures alone was 216,800 +/- 66,480 dpm [3H]uridine.2h-1 X 10(6) cells-1 (mean +/- SD) compared to 98,390 +/- 23,595 in rat Sertoli cells which had been co-cultured with germ cells of fraction 3 for 24 h (P less than 0.01). By contrast, RNA synthesis in Sertoli cell monolayers prepared from immature pigs were unaffected by co-culture with rat germ cells. A similar inhibitory effect of germ cells was observed in rat Sertoli cells stimulated with FSH or testosterone. Culture medium, conditioned by 20 h culture of a fresh preparation of rat spermatogenic cells of fraction 3, was active in inducing the inhibitory effect on RNA synthesis in rat Sertoli cells. Co-culture of rat Sertoli cells with germ cells of this fraction also decreased the incorporative of [3H]thymidine into DNA in rat Sertoli cells, from 9061 +/- 3339 to 4766 +/- 526 dpm.2h-1 X 10(6) cells-1 (P less than 0.01), but no such change was found in pig Sertoli cells. A different spermatogenic cell preparation, partially deprived of pachytene spermatocytes (fraction 5), stimulated rat Sertoli cell DNA synthesis (Sertoli alone 7833 +/- 2550, Sertoli cells which had been in co-culture with germ cells of fraction 5, 13,300 +/- 2279 dpm.2h-1 X 10(6) cells-1, P less than 0.05). These inhibitory actions of some germ cells on Sertoli cells were observed together with the previously reported simultaneous stimulatory effect of Sertoli cells on germ cells. These Sertoli cell-germ cell interactions of detected in culture may represent regulatory influences operating in vivo.     

Age-dependent Sertoli cell responsiveness to germ cells in vitro

This study investigated the effect of germ cells (greater than 80% mid- and late-pachytene spermatocytes) on the secretion of androgen binding protein (ABP) and transferrin by monolayer cultures of Sertoli cells isolated from rats aged 10, 18 or 26 days. There was an age-dependent increase in secretion of ABP and transferrin. Treatment of the Sertoli cell monolayers with hypotonic buffer to remove residual germ cells reduced this increase significantly. On the other hand, addition of germ cells to hypotonic-treated Sertoli cell monolayers increased both basal and FSH + testosterone-stimulated ABP and transferrin secretion at all three ages, although Sertoli cells from 10-day-old animals showed the greatest response. Moreover, addition of germ cells reduced responsiveness to FSH + testosterone in Sertoli cell monolayers obtained from rats aged 18 or 26 days. In monolayers obtained from 10-day-old rats, the opposite effect was noted in the case of ABP secretion. The stimulatory effect of germ cells on ABP and transferrin secretion was proportional to their number, and was reversed 48 h after the germ cells added previously were removed by hypotonic treatment. Whereas the reversal was complete with cultures of Sertoli cells isolated from 18- and 26-day-old rats, approximately 40% of the stimulatory effect remained after removal of germ cells from cultures from the 10-day-old age group. Adhesion of germ cells to Sertoli cell monolayers was also found to be age-dependent, with the largest proportion of added germ cells adhering to Sertoli cells isolated at 18 and 26 days of age. It is concluded that germ cells can significantly and differentially modulate the basal and hormone-stimulated secretory activity of Sertoli cells in vitro and that Sertoli cell responsiveness to germ cells (pachytene spermatocytes) is age-dependent and seems to appear early during the maturation process, before these germ cells appear in the testis.     

Germ cells and post-natal development of testicular function: in vitro studies]

Studies in recent years have clearly established that, in addition to the well known endocrine regulation by gonadotrophin hormones, spermatogenesis is under the modulatory control of a complex set of paracrine regulators. Whereas the role of Leydig cells (testosterone) and of Sertoli cells (nurce cells of germ cells) in spermatogenesis has focused most of the attention, until recently little was known about the contribution of germ cells in the spermatogenetic process. This was the aim of the present experiments. We have used, in vitro, 3 complementary approaches; 1) we measured the influence of the removal of germ cells contaminating Sertoli cell cultures by a hypotonic treatment; 2) in coculture, we examined to what extend isolated germ cells could affect Sertoli cell function; 3) we investigated the effects of germ cell conditioned media on Sertoli cell cultures. Our results indicate that germ cells are able to modulate Sertoli cell function in vitro. This germ cell influence varies according to: 1) the germ cell fraction tested (pachytene spermatocytes, early spermatids or cytoplast from elongated spermatids/residual bodies); 2) the parameter of Sertoli cell function studied (inhibition of oestradiol; stimulation of androgen-binding protein, transferrin...); 3) the age of the Sertoli cell donors; 4) the hormonal environment (+/- FSH). Furthermore we wave demonstrated that germ cell effects were partly at least mediated via proteinaceous factor(s) detected in germ cell spent media. Taking into account previous in vivo studies and these in vitro results, we have hypothesized that germ cells, in conjunction with hormones (LH, FSH, testosterone) play an important role in the ontogenesis of Sertoli cells and therefore in spermatogenesis.     

17 Alpha-oestradiol and 17 beta-oestradiol do not affect basal and follicle-stimulating hormone-stimulated inhibin B secretion by highly purified rat Sertoli cells

The effects of 17 alpha-oestradiol and 17 beta-oestradiol on basal and follicle-stimulating hormone (FSH)-stimulated inhibin B secretion by rat Sertoli cells were studied. Sertoli cells were isolated and cultivated from testes of 18-day-old Wistar rats in the presence and absence of FSH and different doses of oestrogens. On day 4 of culture, secreted inhibin was measured by enzyme-linked immunosorbent assay. Neither 17 alpha-oestradiol nor 17 beta-oestradiol had any effect on the secreted inhibin level in either the presence or absence of FSH. It is concluded that these oestradiols do not play an essential role in regulatory processes involving inhibin or FSH.     

Regulatory influence of germ cells on sertoli cell function in the pre-pubertal rat after acute irradiation of the testis

While germ cell regulation of Sertoli cells has been extensively explored in adult rats in vivo, in contrast, very little is known about germ cell influence on Sertoli cell function at the time when spermatogenesis begins and develops. In the present study various Sertoli cell parameters (number, testicular androgen binding protein (ABP) and testin, serum inhibin-B and, indirectly, follicle-stimulating hormone (FSH)) were investigated after the exposure of 19-day-old rats to a low dose of 3 Grays of gamma-rays. Differentiated spermatogonia were the primary testicular targets of the gamma-rays, which resulted in progressive maturation depletion, sequentially and reversibly affecting all germ cell classes. Testicular weight declined to a nadir when pachytene spermatocytes and spermatids were depleted from the seminiferous epithelium and complete or near complete recovery of spermatogenesis and testicular weight was observed at the end of the experiment. Blood levels of FSH and ABP were normal during the first 11 days after irradiation, when spermatogonia and early spermatocytes were depleted. While the number of Sertoli cells was not significantly affected by the irradiation, from days 11-66 after gamma-irradiation, ABP production declined and FSH levels increased when pachytene spermatocytes and spermatids were depleted and the recovery of these parameters was only observed when spermatogenesis was fully restored. Comparison of the pattern of change in serum levels of inhibin-B and testicular levels of testin and of germ cell numbers strongly suggest a relationship between the disappearance of spermatocytes and spermatids from the seminiferous epithelium and the decrease in levels of inhibin-B and increase in levels of testin from 7 to 36 days post-irradiation. Levels of testin and inhibin-B were restored before spermatogenesis had totally returned to normal. In conclusion, this in vivo study shows that pre-pubertal Sertoli cell function is under the complex control of various germ cell classes. This control presents clear differences when compared with that previously observed in adult animals and depends on the Sertoli cell parameter of interest, as well as on the germ cell type.     

Quantitative evaluation of the maintenance and development of spermatocytes and round spermatids in cultured tubule fragments from immature rat testis

Maintenance and development of spermatocytes and round spermatids was studied in an in-vitro incubation system. This system consisted of open tubule fragments from 26-day-old rat testes, obtained after collagenase treatment. The tubule fragments contained Sertoli cells and spermatogenic cells up to and including a small number of early round spermatids. The number of primary spermatocytes and round spermatids in the tubule fragments was estimated using flow-cytometric analysis, immediately after isolation and after 72 h of incubation. In addition, the activity of LDH-C4 in the tubule fragments was measured. After 72 h of incubation, the percentage of spermatocytes was reduced by 70-80%, but the percentage of spermatids was doubled. The total LDH-C4 activity per well was increased 2-3-fold during 72 h of incubation of the fragments. A modest improvement of the culture results was observed when a combination of FSH, insulin, retinol and testosterone was added to the medium. LDH-C4 activity was investigated to see whether it could be used as a quantitative marker of isolated and cultured spermatocytes and spermatids. It was observed that LDH-C4 activity per cell was decreased when spermatocytes and spermatids were isolated and/or incubated at 4 degrees C. However, the cellular enzyme activity returned to control values during subsequent incubation of the cells at 32 degrees C, either in the absence or presence of a protein synthesis inhibitor. Cellular LDH-C4 activity may be influenced not only by temperature, but possibly also by other cell isolation conditions. It is concluded that LDH-C4 activity may not be a reliable quantitative marker for the presence of spermatocytes and spermatids in culture, but should be used in combination with other analytical methods such as DNA estimation and DNA flow cytometry.     

Hormonal regulation of inhibin B secretion by immature rat sertoli cells in vitro: possible use as a bioassay for estrogen detection

The influences of follicle-stimulating hormone (FSH), gonadal steroids, and culture time were studied in relation to inhibin B production by Sertoli cells of immature rats cultured in vitro. Sertoli cell-enriched cultures were established from 18-day-old rats and were maintained in medium supplemented with insulin, transferrin, and epidermal growth factor at 34 degrees C. A recently developed ELISA for the measurement of inhibin B was used to assess the effects of recombinant human FSH (rh FSH), testosterone (T), and estradiol (E2) on inhibin B production and accumulation in the culture media of Sertoli cell-enriched cultures and to optimize the cell culture system to serve as a bioassay for the detection and quantification of estrogens and estrogenlike substances. Prolonging the incubation time (24, 48, or 72 hours) of Sertoli cells with control medium without rh FSH, T, or E2 resulted in a time-dependent increase of inhibin B production. Incubation with rh FSH (1, 2.5, 5, or 10 U/L) caused a dose- and time-dependent increase of inhibin B production by Sertoli cells (but not by cultured Leydig cells), reaching a plateau at 5 U/L rh FSH. Addition of T in concentrations of 2.88, 5, or 50 ng/ml to medium without rh FSH and E2 significantly lowered the daily production rate of inhibin B (P < 0.05). In contrast, addition of E2 (0.01 and 0.1 ng/ml) caused a dose-responsive increase in inhibin B production after 24 and 48 hours. The relative increment of inhibin B production induced by E2 was maximal after 24 hours in the presence of 2.5 U/L rh FSH (acting synergistically) and in the absence of T. When these conditions are implemented, the Sertoli cell culture system may serve as a bioassay for estrogenic substances, and it may reflect the possibly harmful effect they may have on spermatogenesis.     

Age as a factor influencing the power and sensitivity of experiments for assessing body weight, testis size, and spermatogenesis in rats

Data from 25 rats aged 60, 150, or 240 days (n = 75) were used to determine the number of observations per rat and or rats per treatment group needed to provide future experiments of known power and sensitivity. End points examined included body weight, testicular weight, number of resistant elongated spermatids, yields of germ cells from their younger progenitors, and germ cell:Sertoli cell ratios. Requirements differed in relation to the experimental power and sensitivity desired. However, the replication needed for detecting equivalent treatment effects on paired testes weight or the number of spermatids per gram of testis increased with age, and was twice as great for 240- vs 60-day-old rats. In contrast, approximately twice as many 60-day-old rats were required for detecting treatment effects on the number of spermatids per Sertoli cell than when 150- or 240-day-old rats were used.     

Phosphorylation of mitogen-activated protein kinase 8 (MAPK8) is associated with germ cell apoptosis and redistribution of the Bcl2-modifying factor (BMF)

Successful spermatogenesis requires that germ cells remain in physical contact with Sertoli cells until spermiation. Previous studies have shown that the Bcl2-modifying factor (BMF) is a proapoptotic protein found in many epithelial cells which, when phosphorylated by the active form of mitogen-activated protein kinase 8 (p-MAPK8), initiates apoptosis in response to loss of adhesion of the cells to their basal lamina. Based on this, we hypothesized that p-MAPK8 and BMF may play important roles in the apoptotic death of testicular germ cells in response to their detachment from Sertoli cells. Immunohistochemical analysis of the normal rat testis revealed p-MAPK8 expression in spermatocytes and elongated spermatids but not in round spermatids. This localization was opposite to that of BMF, which is expressed in round spermatids but not in spermatocytes or elongated spermatids. When freshly isolated germ cells were cultured in the absence of Sertoli cells, a condition in which there was widespread germ cell apoptosis, an increase in p-MAPK8 relative to overall MAPK8 protein, was seen by Western blot analysis. Additionally, immunocytochemical analysis showed an increase in immunoreactive p-MAPK8 in round spermatids and spermatocytes in association with BMF expression. From these correlative data, we propose that the activation of MAPK8 and redistribution of BMF may be integrally involved in the mechanism by which specific germ cells undergo programmed cell death in response to their detachment from Sertoli cells.     

The Sertoli-spermatid junctional complex adhesion strength is affected in vitro by adjudin

The actin-based cell-cell adherens junction (AJ) between the Sertoli cell and the germ cell in the mammalian testis is important not only in mechanical adhesion of the cells, but in the morphogenesis and differentiation of the germ cells. The Sertoli ectoplasmic specialization (ES), a specialized type of AJ, is associated with Sertoli-spermatid binding and is important in cell-cell adhesion in the seminiferous epithelium. Abnormal or absent Sertoli ESs have been associated with step-8 spermatid sloughing and oligospermia in conditions associated with reduced fertility potential. The reproductive hormones, follicle stimulating hormone (FSH), and testosterone (T) have also been shown to play a role in the regulation of binding of spermatids at the Sertoli-spermatid junctional complex (STJC). Adjudin [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide] is a potential male contraceptive and is thought to exhibit its contraceptive effects by interrupting the STJC. It has been shown that this compound induces reversible germ cell loss from the seminiferous epithelium, particularly elongating/elongate/round spermatids and spermatocytes. Using a micropipette pressure transducing system (MPTS) to measure the force needed to detach step-8 spermatids from Sertoli cells, this study examined the strength of the STJC in Sertoli-spermatid cocultures in the presence of Adjudin (1 ng/mL, 50 ng/mL, 125 ng/mL, or 500 ng/mL in EtOH) and hormones [FSH (0.1 micro g/mL, NIDDK-oFSH-20, AFP7028D, 175 x NIH-FSH-S1), T (100 nM)] to optimize in vitro binding. The average forces required to detach the spermatids from the underlying Sertoli cells in the presence of 1 ng/mL, 50 ng/mL, 125 ng/mL, and 500 ng/mL Adjudin were 18.2 x 10(-10) pN, 14.3 x 10(-10) pN, 7.74 x 10(-10) pN, and 6.51 x 10(-10) pN, respectively. The average force required to detach step-8 spermatids in the presence of vehicle only (control) was 19.0 x 10(-10) pN. A significant difference for Adjudin concentrations at or above 125 ng/mL was determined by one-way ANOVA (P < .05). These data confirm that Adjudin is effective in reducing the strength of the STJC, identifying Adjudin as a potential contraceptive agent in the male by inducing spermatid sloughing and therefore oligospermia.     

Effect of cryptorchidism and orchidopexy on inhibin secretion by rat Sertoli cells

The present study explored the effects of experimental bilateral cryptorchidism (of 21-, 28-, and 35-days duration) and orchidopexy (14 and 42 days) in the adult rat on the secretion of inhibin by cultures of isolated Sertoli cells. Changes in serum levels of gonadotropins, testis weight, and spermatogenesis also were assessed to verify the effectiveness of the surgical procedures. Cryptorchidy resulted in a progressive decline in testicular weight and a loss of germ cells, associated with increasing serum levels of FSH and LH. Inhibin secretion in vitro became nondetectable by 28 days after surgery. At 42 days after orchidopexy, spermatogenesis showed qualitative recovery, with a small increase in testes weight. Levels of LH in the circulation declined, but only to twice the intact control levels. However, inhibin secretion and serum FSH levels returned to nearly normal values. These results indicate that bilateral cryptorchidism severely impairs the secretion of inhibin and possibly other Sertoli cell functions which may account, at least partly, for the increase in circulating FSH levels and the arrest of spermatogenesis. The effects of cryptorchidism on these parameters can be reversed to a large degree by orchidopexy.     

Evidence that in a physiological setting Sertoli cell number is the major determinant of circulating concentrations of inhibin B in the adult male rhesus monkey (Macaca mulatta).

The relationship between changes in Sertoli cell number and function and changes in circulating inhibin B concentrations was investigated following unilateral orchidectomy (UO) in the adult rhesus monkey. As expected, the 50% loss in Sertoli cells resulting from UO on day 0 was associated with a rapid and corresponding decline in plasma concentrations of inhibin B. The decrease in inhibin B levels was sustained until the remaining testis was removed on day 44, at which time a compensatory 50% increase (P < 0.05) in the number of round spermatids was evident in the absence of a change in Sertoli cell number. Moreover, Sertoli cell number and inhibin B levels among individual monkeys were highly correlated (r2 = 0.65, P < 0.002). Round spermatid number and inhibin B, however, were poorly correlated (r2 = 0.37, P < 0.04). These findings indicate that, in a physiological setting where the negative feedback control system governing the adult primate testis is operational, Sertoli cell number, rather than function, is the primary determinant of circulating inhibin B levels.     

Construction and preliminary characterization of a series of mouse and rat testis cDNA libraries.

We have constructed a series of 23 cDNA libraries from mouse and rat testicular cells. These include libraries made from whole, intact adult testes; seminiferous tubule cells from adult testes; combined populations of primary spermatocytes from 18-day-old mouse testes; and isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, leptotene plus zygotene spermatocytes, juvenile pachytene spermatocytes, adult pachytene spermatocytes, round spermatids, Sertoli cells from 6-, 8-, 17-, and 18-20-day-old mice, and peritubular cells from 18-20 day old mice, all recovered from outbred white Swiss (CD-1) mice. We also constructed libraries from whole adult testes from five other lines of mice: C57 Bl6/J, C3 HEB, BDF-1, Balb/c, and 129 Sv. Finally, there are two libraries made from populations of Sertoli cells and peritubular cells isolated from testes of 20-day-old Sprague-Dawley rats. Enzymatic dissociation, followed by gradient separation or plating/lysing techniques, was used to prepare populations of specific cell types in purities of 85-98%. cDNAs were synthesized from poly A+ mRNA primed with oligo dT and unidirectionally cloned into the lambda Uni-Zap XR expression vector from Stratagene. Primary titers ranged from 2.1 x 10(5) to 2.9 x 10(8) plaque-forming units, and insert sizes averaged 1.0-1.2 kb. These libraries have been amplified once and submitted to the American Type Culture Collection (ATCC) for distribution to interested investigators. ATCC accession numbers are provided.     

Inhibin B levels in plasma of the male rat from birth to adulthood: effect of experimental manipulation of Sertoli cell number

Sertoli cells undergo important changes in their number and function at different ages in the rat and may be the primary source of circulating inhibin B. The aims of this study were 1) to establish the profile of inhibin B levels from birth to adulthood in normal rats and 2) to identify whether experimental manipulation of Sertoli cell numbers was able to alter this profile. Levels of inhibin B, measured by a specific two-site assay, increased fivefold in normal Wistar rats between day 3 and days 10-15, plateaued, and then declined in late puberty to reach adult levels which were approximately 60% of those observed on days 10-15. The increase in inhibin B levels in the neonatal period coincided with the period of Sertoli cell multiplication as indicated by incorporation of bromodeoxyuridine. Neonatal treatment of rats with a GnRH antagonist (GnRHa) reduced Sertoli cell number and adult testis weight by 48% and significantly reduced plasma levels of inhibin B at all ages through to adulthood. Induction of neonatal hypothyroidism in Sprague-Dawley rats by administration of propylthiouracil (PTU) up to day 25 of age increased final testis weight by 41% (indicative of increased Sertoli cell numbers) and resulted in elevation of plasma levels of inhibin B at all ages beyond 7 days of age. The degree of change in inhibin B levels in adult rats in the two experimental treatment groups was approximately proportional to the change in final testis weight. Plasma follicle-stimulating hormone (FSH) showed changes opposite to inhibin B, with levels being lowered in PTU-treated rats and elevated (beyond day 25) in GnRHa-treated animals. The present results suggest that final Sertoli cell number per testis exerts an important effect on the circulating level of inhibin B (and FSH) in the rat. These findings are compared to the emerging data for the human male.     

Effect of testosterone, estradiol-17 beta, and 5 alpha-dihydrotestosterone on inhibin production by sertoli cells in culture

Inhibin activity in charcoal-extracted spent culture medium of Sertoli cells in Primary culture (CSCCM) was monitored by following the temporal inhibition of serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in normal adult male rats. Subcutaneous injection of CSCCM reduced LH and FSH levels within 15 min and maintained the inhibition up to 10 hr. No selective suppression of FSH was observed. The model was used to assess inhibin activity in CSCCM from Sertoli cell cultures incubated with 5 alpha-dihydrotestosterone (DHT), testosterone (T), and estradiol -17 beta (E2) (5 micrograms/ml) for 24 hr. CSCCM from T and E2 treated groups suppressed gonadotropins while the group exposed to DHT failed to elicit any inhibition. DHT seems to inhibit the release of inhibin by Sertoli Cells.     

The effect of selective destruction and regeneration of rat Leydig cells on the intratesticular distribution of testosterone and morphology of the seminiferous epithelium

This study was designed to explore the relationship between the intratesticular distribution of testosterone and spermatogenesis by completely destroying the Leydig cells of mature male rats with injection of a single i.p. dose of ethane dimethanesulphonate. After such treatment, testosterone levels in serum, testicular interstitial fluid, seminiferous tubules, and whole testis declined significantly 6 to 24 hours after injection and fell below assay detection limits between 3 and 7 days. At 3 and 7 days, serum LH and FSH levels rose significantly and remained elevated up to 4 and 6 weeks, respectively, in comparison with vehicle-treated controls. Leydig cells disappeared from the interstitium by day 3, but between 2 and 4 weeks postinjection a new generation of fetal-like Leydig cells repopulated the testicular interstitium and, during weeks 6 to 10, were transformed into, or replaced by, Leydig cells with an adult type of morphology. Histologic examination of the seminiferous tubules showed progressive disruption of spermatogenesis between 3 and 14 days post-ethane dimethanesulphonate. The first histologic sign of spermatogenic damage was noted at day 3, with the occurrence of stage-specific degenerating pachytene primary spermatocytes at stages VII to VIII of the spermatogenic cycle. On day 7, these cells and degenerating round, or step 19, spermatids often were observed during stages VII to XI, although qualitatively normal spermatogenesis also was seen in these and all other stages of the cycle. Maximum impairment of spermatogenesis occurred 2 weeks post-ethane dimethane sulphonate, at which time the tubules commonly lacked one or more germ cell generations or, alternatively, showed accumulation of lipid inclusions, extracellular spaces, and variable numbers of degenerating germ cells. Following repopulation of the testis by Leydig cells during weeks 3 and 4, spermatogenesis recovered. By 10 weeks after treatment, qualitatively normal spermatogenesis was seen in the great majority of seminiferous tubules, although a few tubules still remained in which the germ cell complement was severely reduced, and contained only Sertoli cells and spermatogonia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential regulation of inhibin subunits by germ cells in human testes

Inhibin B is comprised of two dissimilar disulfide-linked subunits, termed alpha and betaB, and is physiologically more important than inhibin A in the male. The aim of this study was to investigate testicular expression of inhibin subtypes in infertile men to uncover any interaction between Sertoli cells and germ cells. Ten azoospermic patients with Sertoli cell only syndrome (SCO) and 39 oligozoospermic men were included in this study. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were determined by chemiluminescence assays. The serum concentrations of inhibin B were measured by enzyme-linked immunosorbent assay. Immunohistochemical staining for the alpha-subunit, betaA-subunit, and betaB-subunit of inhibin were performed on testicular biopsy specimens. The results were that serum inhibin B was undetectable in azoospermic men with SCO, while it was 133.8 +/- 82.0 pg/ml in oligozoospermic men. There was little expression of betaA in the testes of any patient. Expression of inhibin alpha and betaB was observed in Sertoli cells. The percentage of Sertoli cells expressing inhibin alpha was similar in azoospermic patients with SCO (55.3% +/- 20.6%) and in oligozoospermic patients (42.8% +/- 30.4%). In contrast, expression of betaB in Sertoli cells of azoospermic patients (24.9% +/- 16.8%) was lower than in oligozoospermic men (43.4% +/- 25.5%: P = 0.0308). There are no significant correlations between testicular expression of inhibin betaB and the serum inhibin B concentrations. The expression of inhibin betaB by Sertoli cells is dependent on the coexistence of spermatogenic activity within these seminiferous tubules, explaining why the level of inhibin B is low in patients with SCO.     

Expression of galectin-3 and its regulation in the testes

Although spermatogenesis is a complex process under hormonal control, which includes mainly follicle stimulating hormone (FSH) and androgens, little is known about the intra-testicular mediators of these hormones. In the present study, galectin-3 (Gal-3) expression has been identified in human, rat and porcine testes where it is under hormonal control. Gal-3 is present in Sertoli cells and appears to be absent in human and (probably) in rat germ cells. Gal-3 expression was evidenced in the testes, in terms of both mRNA and protein (31 kDa). Gal-3 expression in cultured porcine Sertoli cells was shown to be under the positive control of FSH as well as of two cytokines epidermal growth factor (EGF) and tumour necrosis factor-alpha (TNF-alpha). Gal-3 expression in Sertoli cells is also potentially under the control of mature germ cells as an increased expression was observed in adult rat testes depleted in spermatocytes or spermatids. Although the function of testicular Gal-3 remains to be investigated, a potential role of Gal-3 in germ cell survival/regeneration is suggested based on its increased expression 1 month after a transient germ cell death process triggered by 10 days of treatment with the antiandrogen flutamide. Finally, although in the normal human testes, Gal-3 is exclusively located in the Sertoli cell cytoplasm, a nuclear localization is observed in the infertile testes. Together, the present findings have shown that (i) Gal-3 is expressed in the porcine, rat and human Sertoli cells; (ii) Gal-3 is under the positive control of FSH as well as of EGF and TNF-alpha and possibly of adult germ cells. These observations are compatible with a potential pro-survival role of Gal-3 in the testes.     

Evaluation of the relative importance of endocrine and paracrine factors in control of the levels of inhibin in testicular interstitial fluid

This study aimed to identify the role of endocrine (FSH, LH, testosterone) or paracrine (Leydig or germ cell) factors in control of the secretion of inhibin into testicular interstitial fluid (IF). This was done by measuring inhibin and testosterone levels in IF, and serum gonadotrophin and testosterone levels in adult rats following the destruction of Leydig cells with ethane dimethane sulphonate (EDS), alone or in combination with testosterone ester (TE) supplementation at various doses initiated at various times after EDS treatment. The effect of germ cell loss (induced by local testicular heating) on its own or in combination with the above treatments was also assessed. Treatment with EDS led to major increases in the levels of inhibin in IF and of FSH and LH in serum whilst testosterone levels in IF and serum fell to undetectable levels. Supplementation with TE (1-25 mg) for 21 days from the time of EDS treatment failed to prevent the initial (+3 days) increase in IF levels of inhibin but thereafter suppressed inhibin to control levels or lower and grossly suppressed FSH and LH levels, irrespective of whether the dose of TE administered did (25 or 5 mg) or did not (1 mg) prevent major seminiferous tubule damage. Partial regeneration of Leydig cells and normalization of testosterone levels occurred in rats 21 days after treatment with EDS alone but this failed to normalize inhibin and gonadotrophin levels. When supplementation with TE (25 mg) was initiated at 3, 6 or 9 days after EDS treatment, IF levels of inhibin were normalized within 3 days and maintained thereafter in parallel with suppression of serum FSH and LH to below control levels. Seminiferous tubule damage induced by local testicular heating (43 degrees C for 30 min) led to increased IF levels of inhibin 3 and 14 days later, in parallel with increased serum levels of FSH (but not LH). Suppression of FSH to subnormal levels in heat-exposed rats by TE treatment (25 mg) restored IF inhibin to control levels or below, a change which still occurred when Leydig cells were destroyed by EDS treatment. It is concluded that secretion of inhibin via the base of the Sertoli cell into testicular IF is controlled primarily by FSH, although local factors may play a minor role. These findings have important implications regarding the possible paracrine role(s) of inhibin in IF during puberty and in the normal adult testis.